This invention relates to the sterilization and cryopreservation of tissues for storage. The tissues may be harvested from human or animal subjects and are then processed and cryopreserved (frozen) for later implantation. Allograft tissues, including, but not limited to, heart valves and portions of heart valves, aortic roots, aortic walls, connective tissues including fascia and dura, vascular grafts (including arterial, venous, and biological tubes), and orthopedic soft tissues, such as boned- or non-boned tendons or ligaments, are often subjected to cryogenic preservation. In this manner, a ready supply of these valuable tissues can be made available for later implantation into mammals, especially humans. In addition, viable xenograft tissues from transgenic animals or tissues developed from human or non-human cells that may include differentiated cell types, stem cells, or genetically-modified cells of various origins may be appropriately processed, cryopreserved, and stored for later implantation.
In addition to living allograft, xenograft, or bio-engineered tissues, which may be cryopreserved, living tissues may be decellularized to render them acellular. While living tissues are often decellularized before cryopreservation, they can also be decellularized after cryopreservation and storage.
Generally, decellularization involves substantially reducing the living, non-structural constituents within the tissue. This may be achieved by first exposing the tissue to a hypotonic solution to lyse the cells and then subjecting the tissue to a nuclease treatment to degrade nucleic acids. A more detailed discussion of tissue decellularization may be found in U.S. Pub. No. US 2001/0000804 A1, which is incorporated by reference in its entirety, except that in the event of any inconsistent disclosure or definition from the present application, the disclosure or definition herein shall prevail.
Cryopreservation may be a preferred method of preserving living or decellularized tissue for extended storage. By cryopreserving a tissue in a suitable cryoprotectant, it may be possible to reduce the damage to the tissue that can occur during uncontrolled rate freezing, freeze-drying, and glutaraldehyde preservation methods. In contrast to uncontrolled rate freezing and freeze-drying, cryopreservation preferably involves the addition of one or more cryoprotectant containing solution to the tissue followed by a slower, controlled-rate freezing regimen, which can be adjusted to the particular requirements of each tissue to which it is applied. Cryoprotectants can limit cell or tissue damage due to the formation of ice (water) crystals during freezing and thawing.
In general, there are two requirements for the successful cryopreservation of living tissue. First, the harvested tissue should be frozen to a sufficiently low temperature so metabolic activity effectively ceases within the cell, without destroying the cell. Second, the cryopreservation and thawing regimens should have minimal effects on tissue cell viability and limit structural damage to the tissue. When decellularized tissue is cryopreserved, the primary focus is preventing damage to the structure of the extracellular matrix.
Varying cryoprotectant containing solutions have been used to cryogenically preserve biological tissues for later implantation. Some cryopreservation solutions use a combination of dimethylsulfoxide (DMSO) and Fetal Bovine Serum (FBS) with other constituents for tissue preservation. In these solutions, DMSO may provide interference with the ability of water to form ice crystals during freezing and radical scavenging. However, a disadvantage of DMSO based cryopreservation solutions is the unpleasant odor given off from irradiated solutions upon thawing. DMSO containing cryopreservation solutions can also demonstrate toxicity to living tissues above 4° C. This toxicity and the detailed thawing procedures required to limit its adverse affects on heart valves are described in U.S. Pat. No. 4,890,457, for example.
Because Fetal Bovine Serum is animal-derived, its use in cryopreservation solutions may introduce undesirable contaminants. In addition to the risk of contamination by bacteria and viruses, prion contamination is also possible. It is currently believed that Creutzfeldt-Jakob disease or variant bovine spongiform encephalopathy is transmitted through prions. Not only does FBS open the possibility of disease transmission, but due to the limited availability of cows believed free of prion transmitted disease, FBS can only be harvested from a limited number of herds in specific countries.
Unlike tissues sourced from other human individuals (allografts), which are classified by regulatory authorities as tissues intended for transplantation and whose processing includes procedures to reduce the risk of contamination by pathogenic agents, tissues sourced from different species (xenografts) are classified as medical devices and must be terminally sterilized prior to implantation. Sterilization techniques and conditions are preferably selected to provide a high level of assurance against contamination of the tissue with microbes, while limiting damage to the structure and function of the tissue. These same techniques may also reduce the activity or infectivity of other pathogenic agents, such as viruses, thus increasing the safety factor of the implantable device.
Decellularized tissues can be terminally sterilized using a variety of physical or chemical sterilants, or combinations thereof, including gamma radiation, electron-beam radiation, ethylene oxide, peracetic acid, β-propiolactone, povidone-iodine, or UV irradiation in the presence or absence of photosensitizers. Irradiation methods are preferred. One reason for this is that radiation can be administered to the tissue after final packaging, thus eliminating the necessity to accomplish and validate aseptic transfer of the tissue to its final packaging after sterilization.
Ionizing radiation, including gamma and electron-beam radiation, can be effective in killing a variety of microbial and pathogenic organisms, including bacteria, fungi, yeast, mold, mycoplasmas, parasites, and virus, when an appropriate cumulative dose of radiation is applied to the tissue. A more detailed discussion of gamma radiation and its use in the sterilization of preserved tissues may be found in U.S. Pat. No. 5,485,496, incorporated herein by reference in its entirety, except that in the event of any inconsistent disclosure or definition from the present application, the disclosure or definition herein shall prevail. A more detailed description of electron-beam radiation and its use in the sterilization of preserved tissues may be found in U.S. Pat. No. 5,989,498, also incorporated herein by reference in its entirety, except that in the event of any inconsistent disclosure or definition from the present application, the disclosure or definition herein shall prevail.
While ionizing radiation is convenient, different tissues have various degrees of susceptibility to damage by the energetic free-radicals that are generated during irradiation. While many types of damage may occur during irradiation, one of the most common is free-radical initiated cleavage of the primary amino acid sequence of the structural proteins. The effect of this scission is to cause tissue denaturation. Tissue denaturation can occur when the proteins of the tissue undergo conformational changes that disrupt the structure of the tissue. These structural changes can result in a loss of tissue function after implantation.
Tissue treatment with ionizing radiation may also alter tissue characteristics by producing crosslinking between tissue proteins. Crosslinking can occur when additional covalent bonds form between previously unbonded portions of one or more molecules. Energetic free-radicals are common initiators for radical crosslinking reactions.
Free-radicals or radicals are defined as highly energetic atoms or groups that possess an unpaired electron. The ability of radicals to damage tissues is well known. In addition to initiating crosslinking reactions, radicals can act as oxidizing or reducing agents that can further damage the tissue. Radical species may be generated by the irradiation of the tissue itself, the cryopreservation solution, or the packaging. While irradiation can form many radical species, oxygen and hydroxyl radicals are believed the most common species formed in aqueous solutions, such as cryopreservation solutions.